Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Glucose Regulated Protein 94 (GRP94) resides in the endoplasmic reticulum and is a molecular chaperone or stress protein which is a member of the heat shock protein (HSP) 90 family. The family includes the htpG gene in bacteria, HSP82 in yeast, HSP90 α and β in higher eukaryotes and the TRAP1 protein in mitochondria (Buchner, J. 1999. Hsp90 & Co.—a holding for folding. Trends Biochem Sci 24:136). HSP 90 proteins are ligand regulated and participate in the conformational maturation of protein substrates involved in diverse cellular activities ranging from cell signaling to bacterial recognition and immunomodulation. Extensive work in cell culture models show that GRP94 expression is regulated by reduced levels of glucose (Lee, A. S., et al., Transcriptional Regulation of Two Genes Specifically Induced by Glucose Starvation in a Hamster Mutant Fibroblast Cell Line. J. Biol. Chem., 1983. 258: p. 597-603), perturbations of cellular calcium level (Drummond, I. A., et al., Depletion of intracellular calcium stores by calcium ionophore A23187 induces the genes for glucose-regulated proteins in hamster fibroblasts. J. Biol. Chem., 1987. 262(26): p. 12801-5; Little, E. and A. S. Lee, Generation of a mammalian cell line deficient in glucose-regulated protein stress induction through targeted ribozyme driven by a stress-inducible promoter. J. Biol. Chem., 1995. 270(16): p. 9526-34) or the redox potential (Kim, Y. K., K. S. Kim, and A. S. Lee, Regulation of the glucose-regulated protein genes by b-mercaptoethanol requires de novo protein synthesis and correlates with inhibition of protein glycosylation. J. Cell. Physiol., 1987. 133(3): p. 553-559), inhibition of glycosylation, or activation of the unfolded protein response (Gass, J. N., N. M. Gifford, and J. W. Brewer, Activation of an unfolded protein response during differentiation of antibody-secreting B cells. J Biol Chem, 2002. 277(50): p. 49047-54).
Given all the factors that regulate its expression, it is intriguing that GRP94 is absent from the yeast genome, even though yeast cells respond to these stress situations much like mammalian cells. GRP94 is essentially a protein of multi-cellular organisms, but is clearly not necessary for global protein folding in the ER, nor for the secretory process per se. Therefore, the question arises as to whether and for what processes GRP94 is essential.
Tumors generally contain mutated proteins that often are associated with the transformation process itself. The same mutated proteins also make the tumors biochemically distinct. Accordingly such proteins should be recognized as foreign and elicit vigorous immune response by the T cell arm of the immune system (Velders, M. P., H. Schreiber, and W. M. Kast, Active immunization against cancer cells: impediments and advances. Semin Oncol, 1998. 25:69). Yet, despite the natural capabilities of T cells to kill tumor cells, in practice the immune responses to cancer, while detectable, are weak. This is due to the evolution of multiple mechanisms within tumor cells to evade the immune reconnaissance system altogether, or to decrease the “fire power” of T cells.
Srivastava et al., showed that GRP94 within tumors binds peptides (See Tamura, Y., et al., Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science, 1997. 278:117-120), is released from dying cells (See Basu, S., et al., Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway. Int Immunol, 2000. 12:1539-1546) and then is taken up by macrophages and/or dendritic cells (See Binder, R. J., D. K. Han, and P. K. Srivastava, CD91: a receptor for heat shock protein gp96. Nat Immunol, 2000. 1:151-155; Berwin, B., J. P. Hart, S. Rice, C. Gass, S. V. Pizzo, S. R. Post, and C. V. Nicchitta. 2003. Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells. Embo J 22:6127), where the peptide dissociates from GRP94 and is transferred onto class I histocompatibility proteins as described by Tamura, Y., et al. (FIG. 1). Because peptides displayed by class I molecules stimulate primarily CD8+ T cells, this so-called “peptide re-presentation” pathway leads to enhanced killer cell activity against the tumors, shown to be increased 10-100 fold by this pathway, in cultured cells by the inventor's laboratory and in mouse models by Srivastava's laboratory (See Suto, R. and P. K. Srivastava, A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science, 1995. 269:1585-1588 and Blachere, N. E., et al., Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J. Exp. Med., 1997. 186:1315-1322). GRP94 elicits antigen-presenting cell (APC) activation and directs peptides into the cross-presentation pathways of APC through interactions with Toll-like (APC activation) and endocytic (cross-presentation) receptors of APC (Vabulas, R. M., S. Braedel, N. Hilf, H. Singh-Jasuja, S. Herter, P. Ahmad-Nejad, C. J. Kirschning, C. Da Costa, H. G. Rammensee, H. Wagner, and H. Schild. 2002. The endoplasmic reticulum-resident heat shock protein Gp96 activates dendritic cells via the Toll-like receptor 2/4 pathway. J Biol Chem 277:20847).
Very little is known about GRP94 expression during mammalian development, although the processes of differentiation and organogenesis can be considered as involving natural metabolic stress responses. Whether or not the GRP94 is essential during embryonogenesis has not yet been determined.
Based on the foregoing, it is clear that a need exists for further elucidation of the role played by GRP94 and fragments thereof in modulating the immune process. Such information will provide novel GRP94 based therapeutics for the treatment of cancer, viral infections and other metabolic disorders.